Biology subjects

Stephan, G.

Publications and source records attributed to Stephan, G..

3 recordsLinked to original sources

bioRxiv2021

Activation of the adhesion GPCR GPR133 (ADGRD1) by antibodies targeting the N-terminus

We recently demonstrated that GPR133 (ADGRD1), an adhesion G protein-coupled receptor (aGPCR) whose canonical signaling raises cytosolic cAMP, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. We showed that dissociation of autoproteolytically generated N-terminal and C-terminal fragments (NTF and CTF) of GPR133 at the plasma membrane promotes receptor activation and increases signaling. Toward developing biologics modulating GPR133 function, we tested antibodies against the N-terminus of GPR133 for effects on receptor signaling. Treatment of HEK293T cells overexpressing GPR133 with such antibodies increased cAMP levels in a concentration-dependent manner. Analysis of supernatants following antibody treatment revealed complexes of the antibodies with the autoproteolytically cleaved NTF of GPR133. Cells expressing a cleavage-deficient mutant GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage-dependent. The antibody-mediated stimulation of wild-type GPR133, but not the cleavage-deficient H543R mutant, was reproducible in patient-derived GBM cells. These findings provide a paradigm for modulation of GPR133 function with biologics and support the hypothesis that NTF-CTF dissociation promotes receptor activation and signaling.

cell biology↗

bioRxiv2021

Mimicking extracellular matrix-mediated mechano-activation by antibodies to control signaling of the adhesion G protein-coupled receptor GPR126/ADGRG6

The adhesion G protein-coupled receptor (aGPCR) GPR126/ADGRG6 plays an important role in several physiological functions, such as myelination or peripheral nerve repair. This renders the receptor an attractive pharmacological target. GPR126 is a mechano-sensor that translates binding of extracellular matrix (ECM) molecules to its N terminus into a metabotropic intracellular signal. To date, the structural requirements and the character of the forces needed for this ECM-mediated receptor activation are largely unknown. In this study we provide this information by combining classic second messenger detection with single cell atomic force microscopy. We establish a monoclonal antibody targeting the N terminus to stimulate GPR126 and compare it to the activation through its known ECM ligands collagen IV and laminin 211. As each ligand uses a distinct mode of action, the N terminus can be viewed as an allosteric module that can fine-tune receptor activation in a context-specific manner.

molecular biology↗

bioRxiv2020

Dissociation of the intramolecularly cleaved N- and C-terminal fragments of the adhesion G protein-coupled receptor GPR133 (ADGRD1) increases canonical signaling

GPR133 (ADGRD1), an adhesion G protein-coupled receptor (GPCR), is necessary for growth of glioblastoma (GBM), a brain malignancy. The extracellular N-terminus of GPR133 is thought to be autoproteolytically cleaved into an N-terminal and a C-terminal fragment (NTF and CTF). Nevertheless, the role of this cleavage in receptor activation remains unclear. Here, we show that the wild-type (WT) receptor is cleaved after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant (H543R) in patient-derived GBM cultures and HEK293T cells. However, the resulting NTF and CTF remain non-covalently bound until the receptor is trafficked to the plasma membrane, where we find NTF-CTF dissociation. Using a fusion of the hPAR1 receptor N-terminus and the CTF of GPR133, we demonstrate that thrombin-induced cleavage and shedding of the hPAR1 NTF increases receptor signaling. This study supports a model where dissociation of the NTF at the plasma membrane promotes GPR133 activation. Highlights- GPR133 is intramolecularly cleaved in patient-derived GBM cultures - Cleaved GPR133 signals at higher efficacy than the uncleavable GPR133 H543R mutant - The N- and C-terminal fragments (NTF and CTF) of GPR133 dissociate at the plasma membrane - Acute thrombin-induced cleavage of the human PAR1 NTF from the GPR133 CTF increases signaling eTOC BlurbFrenster et al. demonstrate intramolecular cleavage of the adhesion GPCR GPR133 in glioblastoma and HEK293T cells. The resulting N- and C-terminal fragments dissociate at the plasma membrane to increase canonical signaling. The findings suggest dissociation of GPR133s N-terminus at the plasma membrane represents a major mechanism of receptor activation.

cell biology↗